Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supra-normal concentrations supress preretinal neovascularisation. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/ml. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94 % of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5 h, 24 h, 72 h, 7 days, 14 days and 28 days post-injection (n=6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 ml and the vitreous half-life 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available anti-angiogenic therapeutics. Whilst opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the non-pigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate and the turnover in vitreous is approximately 15% per day.